血塞通注射液对OGD/R损伤小鼠脑微血管内皮细胞VEGF通路表达的影响及机制研究

Effects and mechanism of Xuesaitong Injection on expressions of VEGF pathway in mice brain microvascular endothelial cells after OGD/R

目的研究血塞通注射液对氧糖剥夺/复氧(OGD/R)损伤小鼠脑微血管内皮细胞中血管内皮生长因子(VEGF)、VEGFR-2和可溶性Fms样酪氨酸激酶1(sFlt-1)蛋白表达的影响,探究其对VEGF通路表达的影响及其可能的作用机制。方法采用OGD/R方法建立细胞缺血再灌注损伤模型,以CCK-8法检测细胞活力,确定实验最适细胞种板浓度、复氧时间和血塞通注射液浓度。实验分为正常组、造模组和实验组,正常组正常培养,造模组只造模不用药培养,实验组造模后给血塞通注射液培养。以Western blot方法检测三组细胞中VEGF、VEGFR-2和sFlt-1蛋白的表达情况。结果细胞氧糖剥夺6 h后,实验最适细胞种板浓度为2.5×104个/ml、复氧时间为6 h、血塞通注射液浓度为100 mg/L。在该条件下,与正常组比较,造模组和实验组VEGF、VEGFR-2和sFlt-1蛋白的表达均明显升高,差异有统计学意义(P<0.05);与造模组比较,实验组中VEGF和VEGFR-2蛋白的表达均明显升高,差异有统计学意义(P<0.05),sFlt-1蛋白的表达明显降低,差异有统计学意义(P<0.05)。结论血塞通注射液可上调OGD/R损伤的小鼠脑微血管内皮细胞中VEGF通路信号的表达,其上调VEGF通路信号表达的机制可能与sFlt-1蛋白的表达受到抑制有关。

Objective To study the effect of Xuesaitong Injection on the expression of vascular endothelial growth factor(VEGF),VEGFR-2 and soluble Fms-like tyrosine kinase 1(sFlt-1)protein in brain microvascular endothelial cells of mice injured by oxygen glucose deprivation/reoxygenation(OGD/R),and to explore its influence on the expression of VEGF pathway and its possible mechanism.Methods The OGD/R method was used to establish a cell ischemia-reperfusion injury model,and the cell viability was detected by the CCK-8 method to determine the optimal cell seeding concentration,reoxygenation time and Xuesaitong Injection concentration.The experiment was divided into normal group,model building group,and experimental group.The normal group was cultured normally,and the model building group was only cultured without drugs,while the experimental group was cultured with Xuesaitong Injection after the model was built.Western blot method was used to detect the expression of VEGF,VEGFR-2,and sFlt-1 protein in the three groups of cells.Results After six hours of oxygen and glucose deprivation,the optimal cell seeding plate concentration was 2.5×104 cells/ml,the reoxygenation time was six hours,and the concentration of Xuesaitong Injection was 100 mg/L.Under this condition,compared with the normal group,the expression of VEGF,VEGFR-2,and sFlt-1 protein in the model and the experimental group were significantly increased,and the differences were statistically significant(P<0.05);compared with the model,the expression of VEGF and VEGFR-2 protein in the experimental group were significantly increased,and the differences were statistically significant(P<0.05),and the expression of sFlt-1 protein was significantly reduced,and the difference was statistically significant(P<0.05).Conclusion Xuesaitong Injection can up-regulate the expression of VEGF pathway signals in OGD/R-injured mouse brain microvascular endothelial cells.The mechanism of up-regulating VEGF pathway signal expression may be related to the inhibition of sFlt-1 protein expression.